Recently, coherent Raman scattering microscopy (CRSM) has gained a lot of importance and usage for performing chemical imaging of biological/pharmaceutical/food science-related specimen. The advantage of CRSM over traditional Raman microscopy is the higher speed of imaging. Coherent anti-Stokes Raman scattering (CARS), coherent Stokes Raman scattering (CSRS), Raman-induced Kerr-effect scattering (RIKES) and stimulated Raman scattering (SRS) constitute various CRSM techniques.
The technique of CRSM consists of two pulsed (with frequencies ranging typically from 1-100 MHz) optical fields with pulse widths ranging from 100 fs-20 ps of different wavelengths routed through a confocal microscope system and tightly focused on a sample of interest. The beam routing and the focusing optics are made such that the two optical fields overlap spatially and temporally at the sample. For SRS or heterodyned-RIKES imaging, one of the two beams is either intensity modulated or frequency modulated or polarization modulated with a specific frequency Ω, typically in kHz to MHz range, before it interacts with the other optical field in the sample.
For SRS and RIKES imaging, one detects the initially unmodulated light beam and using a lock-in [1-2, 5] or an envelope detection technique [6], the modulation of intensity is extracted and displayed as an image. Due to the interaction with the sample, a third light field, in case of CARS and CSRS, is detected and displayed as an image [7-9]. In all the above CRSM techniques, the signal is strong only when the frequency difference between the incident fields matches a vibrational resonance frequency in the sample.
Among the various coherent Raman imaging techniques, SRS imaging has gained popularity in the recent times because of the absence of non-resonant background in the images. In the following, it is concentrated exclusively on SRS imaging methodology. However, the conclusions derived are directly applicable to CRSM, CARS, CSRS and RIKES imaging. It is simply a matter of using appropriate polarization elements, detection units, and optical filters to extract these signals and these are well known and described in the existing literature.
A typical SRS microscope system 20 is depicted in FIG. 1. Two pulsed laser sources (Laser 1 and Laser 2) generate the so called “Stokes” and the “pump” beams for SRS imaging, respectively (see FIG. 2, window 100, where an intensity is shown over time t). Typically, the laser source with smaller wavelength is designated as the “pump” and the laser with longer wavelength is designated as the “Stokes” beam in the CRSM literature. One of the beams (Laser 1) is amplitude modulated with the help of a modulator 3, e.g. an AOM (acousto-optic modulator) or EOM (electro-optic modulator), and combined collinearly with the second laser source (Laser 2) using a dichroic 4. The modulator 3 is driven by a RF drive 9 providing a RF frequency signal.
The temporal overlap of the two laser sources is ensured with the help of a preferably variable—optical delay stage 5. The combined beams travel through a scanning-device or laser-scanning (confocal) microscope 6 and interact with the sample under test at the focus of a microscope objective (not shown). Behind the sample the beam of Laser 1 is blocked by a blocking filter 11.
Due to the interaction with the sample, the light of Laser 2 acquires a tiny amplitude modulation whose frequency corresponds to the modulation frequency of the light of Laser 1 (see FIG. 2, window 200).
When both the beams hit the sample, the light of the Laser 2 could gain or lose energy depending on its relative (to the light of the Laser 1) wavelength. If the wavelength of Laser 2 is smaller than the wavelength of Laser 1, i.e. λLaser2<λLaser2, there is an intensity loss ISRL, otherwise a gain ISRG.
This miniscule gain or loss in Laser 2 is detected with the help of a sensitive detector 7 and signal extraction electronics 8. The latter electronic unit can comprise a lock-in amplifier/mixer; the SRS signal is demodulated using the RF drive frequency of the modulator 3 as the local oscillator. The resultant signal is routed for image display, storage or analysis 10.
It has been demonstrated that using appropriate detector and low-noise electronics it is possible to make SRS imaging sensitive enough for real-time imaging (25 frames-per-second) [3].
A lock-in amplifier is an important component of the detection electronics required for extraction of modulated SRS signals. Although the SRS detection has been implemented using envelope detection electronics [6], use of a lock-in detection provides flexible detection bandwidth (implies, varied laser scan frequencies) and higher signal linearity.
A basic lock-in amplifier 300, as shown in FIG. 3, comprises an electronic mixer 301 which takes as its input an amplitude modulated signal s and a reference clock signal r. It provides as an output the demodulated signal of s at the reference frequency provided by r. In addition, commercially available lock-in amplifiers can provide some amount of signal amplification 303 and filtering 302 at the input and/or output ports. The frequency bandwidth of the extracted signal o depends on the bandwidth setting BW of the filter 302 at the output stage of the mixer. This bandwidth setting typically ranges from a few tenths of Hz to multiple MHz (or equivalently, a few tens of seconds to a few nanoseconds).
In an SRS imaging session, one usually changes various image acquisition parameters to obtain a good image or an image which comprises the part/parts of the imaged object which is/are of interest for the user of the microscope. For example, since the Raman signals are much weaker in the fingerprint-region (500 cm−1-1800 cm−1), one would need to increase the pixel dwell-time and perform slow scanning for obtaining good SRS image. Other acquisition parameters which could be modified based on the imaging requirements include laser-scan/stage-scan speed, objective magnification, objective numerical aperture, image scan format (pixel size), image scan width (physical image size), pixel dwell time, zoom, etc. Changing any of these values would lead to a different bandwidth of the modulated signal. If the lock-in time-constant is fixed, varying these parameters could deteriorate image quality.